cDNA cloning, genomic structure and chromosomal localization of the human BUP-1 gene encoding beta-ureidopropionase.

A full-length cDNA clone encoding human beta-ureidopropionase was isolated. A 1152-nucleotide open reading frame which corresponds to a protein of 384 amino acids with a calculated molecular weight of 43? omitted?158 Da, surrounded by a 5'-untranslated region of 61 nucleotides and a 3'-untranslated region of 277 nucleotides was identified. The protein showed 91% similarity with the translation product of the rat beta-ureidopropionase cDNA. Expression of the human cDNA in an Escherichia coli and eukaryotic COS-7 expression system revealed a very high beta-ureidopropionase enzymatic activity, thus confirming the identity of the cDNA. Since human EST libraries from brain, liver, kidney and heart contained partial beta-ureidopropionase cDNAs, the enzyme seems to be expressed in these tissues, in agreement with the expression profile of this enzyme in rat. Using the human cDNA as a probe a genomic P1 clone could be isolated containing the complete human beta-ureidopropionase gene. The gene consist of 11 exons spanning approximately 20 kB of genomic DNA. Fluorescence in situ hydridization localized the human beta-ureidopropionase gene to 22q11.2.     

Structure and Methylation-Based Silencing of a Gene (DBCCR1) within a Candidate Bladder Cancer Tumor Suppressor Region at 9q32–q33

Loss of heterozygosity (LOH) on chromosome 9q is the most frequent genetic alteration in transitional cell carcinoma (TCC) of the bladder, indicating the presence of one or more relevant tumor suppressor genes. We previously mapped one of these putative tumor suppressor loci to 9q32–q33 and localized the candidate region within a single YAC 840 kb in size. This locus has been designatedDBC1(for deleted in bladder cancer gene 1). We have identified a novel gene,DBCCR1,in this candidate region by searching for expressed sequence tags (ESTs) that map to YACs spanning the region. Database searching using the entireDBCCR1cDNA sequence identified several human ESTs and a few homologous mouse ESTs. However, the predicted 761-amino-acid sequence had no significant homology to known protein sequences. Mutation analysis of the coding region and Southern blot analysis detected neither somatic mutations nor gross genetic alterations in primary TCCs. AlthoughDBCCR1was expressed in multiple normal human tissues including urothelium, mRNA expression was absent in 5 of 10 (50%) bladder cancer cell lines. Methylation analysis of the CpG island at the 5′ region of the gene and the induction ofde novoexpression by a demethylating agent indicated that this island might be a frequent target for hypermethylation and that hypermethylation-based silencing of the gene occurs in TCC. These findings makeDBCCR1a good candidate forDBC1.     

Lipophilins: human peptides homologous to rat prostatein

Lipophilin components A, B and C are human homologues of prostatein, the major secreted protein of rat prostate. This report describes their cDNA sequences, tissue expression and chromosomal localization. Lipophilin gene products were widely expressed in normal tissues, especially in endocrine-responsive organs. The gene for lipophilin C (also called mammaglobin b) is located on chromosome 11q12-q13.1, near the mammaglobin gene, a homologue overexpressed in many breast cancers. The lipophilin B gene resides on chromosome 10q23, a region deleted in many tumors, and the lipophilin A gene is on chromosome 15q12-q13.     

Striking differences of LDL receptor-related protein 1B expression in mouse and human

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a member of the expanding LDL receptor family, and is closely related to LRP. It was discovered as a putative tumor suppressor, and is frequently inactivated in human malignant tissues. However, the expression pattern of LRP1B in normal human tissues was unclear. In the present study, we analyzed LRP1B expression in normal mouse and human tissues. By using RT-PCR, we found that, while mouse LRP1B expression is mostly restricted to the brain, human LRP1B expression is more widespread with highest expression levels detected in the brain, adrenal gland, salivary gland, and testis. Although mouse LRP1B expresses in the forms of both full-length receptor tail and an alternatively spliced form lacking a 33-amino acid insert, human LRP1B is expressed exclusively in the form of full-length receptor tail. Finally, we found that, unlike mouse LRP1B, human LRP1B is cleaved by furin. Taken together, these data demonstrate that there are striking differences between LRP1B expression in mouse and human tissues. The broader expression pattern of LRP1B in human tissues suggests that this putative tumor suppressor may play roles in several types of human cancer.     

Identification and Characterization of the Human Ortholog of Rat STXBP1, a Protein Implicated in Vesicle Trafficking and Neurotransmitter Release

In a screen designed to identify genes expressed preferentially in retina, we identified a cDNA encoding the human ortholog of rat STXBP1 (n-Sec1, Munc-18-1, rbSec1), a protein implicated in vesicle trafficking and neurotransmitter release. This protein also has similarity toDrosophilaRop (64% aa identity) andCaenorhabditis elegansUNC-18 (58% aa identity). The major human cDNA encodes a protein of 594 amino acids which has 100% amino acid identity with its rat and murine counterparts. Additionally, there is an alternative splice form in humans, arising from the inclusion of an additional exon, which encodes a protein of 603 amino acids and is also 100% identical to the corresponding rat isoform. We found expression of the shorter cDNA in all tissues and cell lines we examined with highest levels in retina and cerebellum. By RT-PCR analysis, we found expression of the longer cDNA in neural tissues only. We mapped the structural gene to 9q34.1, a region without obvious candidate phenotypes. However, due to its evolutionary conservation and abundant expression in retina and brain, STXBP1 should be considered a candidate gene for retinal and/or neural disorders mapping to 9q34.1.     

Mouse and human GTPBP2, newly identified members of the GP-1 family of GTPase

We earlier identified the GTPBP1 gene which encodes a putative GTPase structurally related to peptidyl elongation factors. This finding was the result of a search for genes, the expression of which is induced by interferon-gamma in a macrophage cell line, THP-1. In the current study, we probed the expressed sequence tag database with the deduced amino acid sequence of GTPBP1 to search for partial cDNA clones homologous to GTPBP1. We used one of the partial cDNA clones to screen a mouse brain cDNA library and identified a novel gene, mouse GTPBP2, encoding a protein consisting of 582 amino acids and carrying GTP-binding motifs. The deduced amino acid sequence of mouse GTPBP2 revealed 44.2% similarity to mouse GTPBP1. We also cloned a human homologue of this gene from a cDNA library of the human T cell line, Jurkat. GTPBP2 protein was found highly conserved between human and mouse (over 99% identical), thereby suggesting a fundamental role of this molecule across species. On Northern blot analysis of various mouse tissues, GTPBP2 mRNA was detected in brain, thymus, kidney and skeletal muscle, but was scarce in liver. Level of expression of GTPBP2 mRNA was enhanced by interferon-gamma in THP-1 cells, HeLa cells, and thioglycollate-elicited mouse peritoneal macrophages. In addition, we determined the chromosomal localization of GTPBP1 and GTPBP2 genes in human and mouse. The GTPBP1 gene was mapped to mouse chromosome 15, region E3, and human chromosome 22q12-13.1, while the GTPBP2 gene is located in mouse chromosome 17, region C-D, and human chromosome 6p21-12.     

EHD1--an EH-domain-containing protein with a specific expression pattern.

A cDNA that is a member of the eps15 homology (EH)-domain-containing family and is expressed differentially in testis was isolated from mouse and human. The corresponding genes map to the centromeric region of mouse chromosome 19 and to the region of conserved synteny on human chromosome 11q13. Northern analysis revealed two RNA species in mouse. In addition to the high levels in testis, expression was noted in kidney, heart, intestine, and brain. In human, three RNA species were evident. The smaller one was predominant in testis, while the largest species was evident in other tissues as well. The predicted protein sequence has an EH domain at its C-terminus, including an EF, a Ca2+ binding motif, and a central coiled-coil structure, as well as a nucleotide binding consensus site at its N-terminus. As such, it is a member of the EH-domain-containing protein family and was designated EHD1 (EH domain-containing 1). In cells in tissue culture, we localized EHD1 as a green fluorescent protein fusion protein, in transferrin-containing, endocytic vesicles. Immunostaining of different adult mouse organs revealed major expression of EHD1 in germ cells in meiosis, in the testes, in adipocytes, and in specific retinal layers. Results of in situ hybridization to whole embryos and immunohistochemical analyses indicated that EHD1 expression was already noted at day 9.5 in the limb buds and pharyngeal arches and at day 10.5 in sclerotomes, at various elements of the branchial apparatus (mandible and hyoid), and in the occipital region. At day 15.5 EHD1 expression peaked in cartilage, preceding hypertrophy and ossification, and at day 17.5 there was no expression in the bones. The EHD1 gene is highly conserved between nematode, Drosophila, mouse, and human. Its predicted protein structure and cellular localization point to the possibility that EHD1 participates in ligand-induced endocytosis.     

Sustained expression of the novel EBV-induced zinc finger gene, ZNFEB, is critical for the transition of B lymphocyte activation to oncogenic growth transformation.

EBV is a human tumor virus that infects and establishes latency in the majority of humans worldwide. In vitro, EBV growth transforms primary B lymphocytes into lymphoblastoid cell lines with high efficiency. We have used cDNA subtraction cloning to identify cellular target genes required for growth transformation and identified a new C(2)H(2) (Krüppel-type) zinc finger gene, ZNF(EB), that is trans-activated early following EBV infection. In this study, we characterize ZNF(EB), including its intronless locus, and human and mouse protein variants. The gene is transiently expressed during normal lymphocyte activation, and its expression is sustained in EBV-positive but not EBV-negative B cell lines. There is limited expression in nonhemopoietic tissues. Its critical role in the growth transformation of B lineage cells is indicated by the abrogation of transformation with antisense strategies. ZNF(EB) maps to chromosome 18q12, a region with mutations in numerous, predominantly hemopoietic malignancies.     

Isolation, tissue distribution and prokaryotic expression of a novel human X-linked gene LHFPL1.

We report here the cloning and characterization of a novel human lipoma HMGIC fusion partner-like 1 (LHFPL1) gene, isolated from human brain cDNA library, and mapped to Xq23 by browsing the UCSC genomic database. LHFPL1 contains an ORF with length of 660bp, encoding a protein with a signal peptide sequence and three transmembrane regions, and its predicted molecular weight is 23.7kDa which coincides with the result of prokaryotic expression. LHFPL1 protein is postulated to be localized at endoplasmic reticulum. RT-PCR amplification in seventen human tissues revealed that LHFPL1 is expressed widely in all tissues, especially highly in lung, thymus, skeleton muscle, colon and ovary. In addition, it was demonstrated that LHFPL1 is also transcribed in six liver tumor cell lines.     

Cloning and expression of a novel retinoblastoma binding protein cDNA,RBBP10

A 2860-bp cDNA was isolated from a human fetal brain cDNA library by high throughput cDNA sequencing, which encodes a putative protein with 186 amino acids. The putative protein shares 90.7% identity with rat pBOG (3403163) and shares 93.4% identity with human RBBP9 (NPA conserved RB binding domain, L × C × E, located between residue 63 and 68 was recognized. Therefore, it was named RBBP10. Mapviewer analysis locates it on human chromosome 20q11.22. RBBP10 spans about 9.6 kb of the genome and consists of six exons and five introns. RT-PCR revealed that the gene was expressed widely in various human tissues, and the expression level is somewhat higher in tumor tissues than in normal tissues. But subsequent sequencing analysis did not found any mutation of this in tumor tissues. The COS 7 cell transfected with the ORF of RBBP10 showed that the protein was distributed both in the cytoplasm and in the nucleus. Our results suggest that RBBP10 is the orthologue of the rat BOG gene (AF025819) and a paralogue of human RBBP9 (AF039564).     

Molecular characterization of a cDNA encoding functional human CLK4 kinase and localization to chromosome 5q35 [correction of 4q35

Phosphorylated serine- and arginine-rich (SR) proteins play an important role in the formation of spliceosomes, possibly controlling the regulation of alternative splicing. Enzymes that phosphorylate the SR proteins belong to the family of CDC2/CDC28-like kinases (CLK). Employing nucleotide sequence comparison of human expressed sequence tag sequences to the murine counterpart, we identified, cloned, and recombinantly expressed the human orthologue to the murine CLK4 cDNA. When fused to glutathione S-transferase, the catalytically active human CLK4 is able to autophosphorylate and to phosphorylate myelin basic protein, but not histone H2B as a substrate. Inspection of mRNA accumulation demonstrated gene expression in all human tissues, with the most prominent abundance in liver, kidney, brain, and heart. Using fluorescence in situ hybridization, the human CLK4 cDNA was localized to band q35 on chromosome 5 [corrected].     

Assignment of the gene for neuroendocrine protein 7B2 (SGNE1 locus) to mouse chromosome region 2[E3-F3] and to human chromosome region 15q11-q15

The gene for 7B2, a protein found in the secretory granules of neural and endocrine cells (gene symbol SGNE1) was localized to the E3-F3 region of mouse chromosome 2 and to the q11-q15 region of human chromosome 15. This was determined by in situ hybridization, using a mouse 7B2 cDNA and an intronic fragment of the corresponding human gene as probes. The respective locations of SGNE1 in the two species correlate with the conservation of loci between these subregions of mouse chromosome 2 and human chromosome 15. Clinically, the human SGNE1 DNA fragment may serve as a molecular probe of this locus in both the Prader-Willi and the Angelman syndromes, which are often accompanied by submicroscopic chromosomal deletions in the 15q11-15q13 region.     

Characterization of two novel KRAB-domain-containing zinc finger genes, ZNF460 and ZNF461, on human chromosome 19q13.1-->q13.4

This study reports the cloning and characterization of two novel human zinc finger protein cDNAs (ZNF460 and ZNF461) from a fetal brain cDNA library. The ZNF460 cDNA is 3,135 bp in length encoding a 562-amino-acid polypeptide and the ZNF461 cDNA is 2,548 bp encoding a 563-amino-acid protein. Both of the proteins contain a KRAB A+B box and eleven C2H2 type zinc finger motifs. ZNF461 shows high similarity with the rat GIOT-1 gene (GIOT1). The ZNF460 gene mapped to 19q13.4 with 3 exons, and ZNF461 mapped to 19q13.1 with 6 exons. Both of the two genes are ubiquitously expressed in normal human tissues and the abundance of the ZNF460 mRNA is relatively low.     

Cell cycle-dependent expression and centrosome localization of a third human aurora/Ipl1-related protein kinase, AIK3

We earlier isolated cDNAs encoding novel human protein kinases AIK and AIK2 sharing high amino acid sequence identities with Drosophila Aurora and Saccharomyces cerevisiae Ipl1 kinases whose mutations cause abnormal chromosome segregation. In the present study, a third human cDNA (AIK3) highly homologous to aurora/IPL1 was isolated, and the nucleotide sequence was determined. This cDNA encodes 309 amino acids with a predicted molecular mass of 35.9 kDa. C-terminal kinase domain of AIK3 protein shares high amino acid sequence identities with those of Aurora/Ipl1 family protein kinases including human AIK, human AIK2, Xenopus pEg2, Drosophila Aurora, and yeast Ipl1, whereas the N-terminal domain of AIK3 protein shares little homology with any other Aurora/Ipl1 family members. AIK3 gene was assigned to human chromosome 19q13.43, which is a frequently deleted or rearranged region in several tumor tissues, by fluorescence in situ hybridization, somatic cell hybrid panel, and radiation hybrid cell panel. Northern blot analyses revealed that AIK3 expression was limited to testis. The expression levels of AIK3 in several cancer cell lines were elevated severalfold compared with normal fibroblasts. In HeLa cells, the endogenous AIK3 protein level is low in G1/S, accumulates during G2/M, and reduces after mitosis. Immunofluorescence studies using a specific antibody have shown that AIK3 is localized to centrosome during mitosis from anaphase to cytokinesis. These results suggest that AIK3 may play a role(s) in centrosome function at later stages of mitosis.     

The alpha1-6-fucosyltransferase gene and its biological significance

GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha1-6-fucosyltransferase (alpha1-6FucT) catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycoproteins. This enzyme was purified from a human fibroblast cell line, porcine brain, a human gastric cancer cell line and human blood platelets. cDNA cloning of porcine and human alpha1-6FucT was performed from a porcine brain and gastric cancer cell cDNA libraries, respectively. Their homology is 92.2% at the nucleotide level and 95.7% at the amino acid level. No putative N-glycosylation sites were found in the predicted amino acid sequence. No homology to other fucosyltransferases such as alpha1-2FucT, alpha1-3FucT and alpha1-4FucT was found except for a region consisting of nine amino acids. The alpha1-6FucT gene is located at chromosome 14q24.3, which is also a different location from other fucosyltransferases reported to date. The alpha1-6FucT gene is the oldest gene family in the phylogenic trees among the nine cloned fucosyltransferase genes. alpha1-6FucT is widely expressed in various rat tissues and the expression of alpha1-6FucT in the liver is enhanced during hepatocarcinogenesis of LEC rats which develop hereditary hepatitis and hepatomas. In cases of human liver diseases, alpha1-6FucT is expressed in both hepatoma tissues and their surrounding tissues with chronic liver disease, but not in the case of normal liver. Serum alpha1-6-fucosylated alpha-fetoprotein (AFP) has been employed for an early diagnosis of patients with hepatoma. The mechanisms by which alpha1-6 fucosylation of AFP occurs in the hepatoma is not due to the up-regulation of alpha1-6FucT alone. Interestingly, when the alpha1-6FucT gene is transfected into Hep3B, a human hepatoma cell line, tumor formation in the liver of nude mice after splenic injection is dramatically suppressed. In this review, we focus on alpha1-6FucT and summarize its properties, gene expression and biological significance.